Psgl-1 modulators and uses thereof

ABSTRACT

The present invention relates to the seminal discovery that P-selectin glycoprotein ligand-1 (PSGL-1) modulates the immune system and immune responses. Specifically, the present invention provides PSGL-1 agonists and antagonists which increase the survival of multifunctional T cells and viral clearance. The present invention further provides methods of treating infectious diseases, cancer and immune and inflammatory diseases and disorders using a PSGL-1 modulator.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 17/086,922, filed Nov. 2, 2020, and is a divisionalapplication of U.S. National Stage application Ser. No. 15/324,262,filed Jan. 5, 2017 (now U.S. Pat. No. 10,858,436, issued Dec. 8, 2020),which claims priority to International Application No. PCT/US15/39586,filed Jul. 8, 2015, and claims benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 62/022,191, filed Jul. 8, 2014, the entirecontents of which are incorporated herein by reference in theirentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under P01 AI046530, R01AI106895-01A1 and P30 CA030199 awarded by the National Institutes ofHealth. The government has certain rights in the invention

FIELD OF THE INVENTION

The present invention relates to generally to antibodies and morespecifically to the use of P-selectin glycoprotein ligand-1 (PSGL-1)modulators and uses thereof to modulate an immune response and treatinfectious diseases, cancer and immune and inflammatory related diseasesand disorders.

BACKGROUND OF THE INVENTION

Immune responses by T lymphocytes are critical for host protectionagainst microbes, but they can become dysfunctional in chronicinfections. With chronic viral infections, such as HIV and hepatitis Band C, T cell responses have a progressive loss of function and many Tcells undergo apoptosis. The remaining T cells are arrested in adysfunctional state, i.e. phenotypic exhaustion. Although isincompletely understood, several underlying mechanisms have beenidentified for T cell exhaustion in the lymphocytic chodomeningititsvirus (LCMV) model of chronic infection with the Clone 13 strain (Cl13).In this model, virus-specific CD8+ T cells are chronically exposed toantigen and type I interferon resulting in an upregulated expression ifimmune inhibitory receptors including PD-1, LAG-3, CD160, and BTLA. Thecells also lose motility, show altered transcriptional regulation, andhave an increased production of the inhibitory cytokines, IL-10 andTGFβ. With time CD8+ T cells lose their proliferative potential,cytotoxic function and ability to produce IL-2, TNF-α and IFN-γ. CD4+ Tcells display similarly altered differentiation with upregulation ofinhibitory receptors and loss of function. Importantly, virus-specificCD4+ T cells can rescue exhausted CD8+ T cells, enabling them to mediateviral clearance. The production of IL-2 and IL-21 by CD4+ T cells isthought to contribute to reversal of the defective CD8+ T cellresponse—implying that key cytokines necessary for appropriate effectordifferentiation become limiting after chronic infection. Therefore aninterplay of several integrated processes combine to disable the immunesystem's ability to eliminate chronic viral infections.

Many cancers including skin, lung, and kidney establish similar statesof immune suppression and share many common features of immunedysfunction as those observed in chronic viral infections including HIV,Hepatitis B and C. These include death of responding T cells, andupregulation of inhibitory receptors including PD-1, Lag-3 and CTLA-4.As such, immunotherapies directed at boosting the critical functions ofanti-tumor T cell responses are necessary to eradicate cancer in thesesettings. It is now evident that chronic viruses and tumors usurp theimmune system by commandeering natural checks that prevent excessiveimmune responses. A major biomedical research problem is how to developtherapies to reverse this immunosuppression and promote tumorelimination.

It is now evident that reversing T cell dysfunction could reestablishimmune responses and achieve disease resolution in a broad range ofclinical settings. Adhesion mechanisms regulate the accumulation of Tcells in both lymphoid and nonlymphoid tissues. Modulating adhesionmolecule expression has been used to influence effector and memory Tcell development and distinguish cells at different stages ofdifferentiation. P-selectin glycoprotein ligand-1 (PSGL-1), a ligand forthe selectin family of receptors, L, E, and P, is highly induced on Tcells after chronic LCMV infection. PSGL-1 is primarily recognized forregulating T cell trafficking into inflamed tissues, and mediating Tcell migration into lymphoid tissues under steady state conditions andafter inflammatory responses. PSGL-1 can also regulate memory T cellhoming to the bone marrow after a response resolves.

Despite new treatments that can greatly improve immune destruction of abroad range of cancers by blocking immune inhibitory receptors (e.g.PD-1/PD-L1, CTLA-4) and by anti-tumor T cells, efficacy is limited tosubset of patients (often <30%). Thus, there is a critical need todevelop new strategies to harness the immune system to achieve effectiveimmunotherapy in the nonresponsive patients.

SUMMARY OF THE INVENTION

The present invention relates to the seminal discovery that P-selectinglycoprotein ligand-1 (PSGL-1) modulates the immune system and immuneresponses. Specifically, the present invention provides PSGL-1 agonistsand antagonists which increase the survival of multifunctional T cellsand viral clearance. The present invention further provides methods oftreating infectious diseases, cancer and immune and inflammatorydiseases and disorders using a PSGL-1 modulator.

In one embodiment, the present invention provides a method of treating aT cell mediated disease or disorder comprising administering aP-selectin glycoprotein ligand-1 (PSGL-1) modulator to a subject in needthereof. In one aspect, the PSGL-1 modulator is an agonist orantagonist. In another aspect, the T cell mediated disease or disorderis an infectious disease, cancer, an autoimmune disorder or aninflammatory disorder. In a specific aspect, the PSGL-1 modulator is anagonist and the T cell mediated disease or disorder is an autoimmune orinflammatory disease or disorder. In certain aspects, the PSGL-1modulator is an antagonist and the T cell mediated disease or disorderis cancer or an infectious disease.

In one aspect, the PSGL-1 modulator is an antibody, a small molecule, aprotein, a fusion protein or a nucleic acid. In a specific aspect, theantibody is a monoclonal antibody, chimeric antibody, human antibody orhumanized antibody. In another aspect, CD4+ dependent CD8+ T cellresponse is increased. In an additional aspect, virus specific T cellsare increased. In another aspect, Treg and DC response is increased. Ina further aspect, expression of FoxP3, IL-10, TGF-β and/or MHC class IIis increased. In an aspect, CD8+ secretion of IFNγ, TNFα and CD107 isincreased. In other aspects, expression of PD-1, BTLA and CD160 isdecreased or increased. In one aspect, expression of CD25 and T-bet isincreased. In another aspect, viral clearance is increased.

In an additional embodiment, the present invention provides for a methodof eliciting a T cell response comprising administering a PSGL-1modulator to a subject in need thereof. In an aspect, the PSGL-1modulator is an agonist or antagonist. In another aspect, the T cellmediated disease or disorder is an infectious disease, cancer, anautoimmune disorder or an inflammatory disorder. In one aspect, thePSGL-1 modulator is an antibody, a small molecule, a protein, a fusionprotein or a nucleic acid. In a specific aspect, the antibody is amonoclonal antibody, chimeric antibody, human antibody or humanizedantibody. In another aspect, CD4+ dependent CD8+ T cell response isincreased. In an additional aspect, virus specific T cells areincreased. In another aspect, Treg and DC response is increased. In afurther aspect, expression of FoxP3, IL-10, TGF-β and/or MHC class II isincreased. In an aspect, CD8+ secretion of IFNγ, TNFα and CD107 isincreased. In other aspects, expression of PD-1, BTLA and CD160 isdecreased or increased. In one aspect, expression of CD25 and T-bet isincreased.

In a further embodiment, the present invention provides a method ofrestoring T cell function comprising administering a P-selectinglycoprotein ligand-1 (PSGL-1) modulator to a subject in need thereof.In an aspect, the PSGL-1 modulator is an agonist or antagonist. Inanother aspect, the T cell mediated disease or disorder is an infectiousdisease, cancer, an autoimmune disorder or an inflammatory disorder. Inone aspect, the PSGL-1 modulator is an antibody, a small molecule, aprotein, a fusion protein or a nucleic acid. In a specific aspect, theantibody is a monoclonal antibody, chimeric antibody, human antibody orhumanized antibody. In another aspect, CD4+ dependent CD8+ T cellresponse is increased. In an additional aspect, virus specific T cellsare increased. In another aspect, Treg and DC response is increased. Ina further aspect, expression of FoxP3, IL-10, TGF-β and/or MHC class IIis increased. In an aspect, CD8+ secretion of IFNγ, TNFα and CD107 isincreased. In other aspects, expression of PD-1, BTLA and CD160 isdecreased or increased. In one aspect, expression of CD25 and T-bet isincreased.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a P-selectin glycoprotein ligand-1 (PSGL-1)modulator and a pharmaceutical carrier. In one aspect, the PSGL-1modulator is an antibody, a small molecule, a protein, a fusion proteinor a nucleic acid. In an additional aspect, the antibody is a monoclonalantibody, chimeric antibody, human antibody or humanized antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show PSGL-1 kinetics and accumulation of virus-specific Tcells in PSGL-1 KO mice after LCMV Clone 13 infection. WT mice arerepresented by black bars or black circles and PSGL-1 KO mice arerepresented by white bars. (A) Mean fluorescence intensity (MFI) ofPSGL-1 levels on GP₃₃₋₄₁+CD8+ T cells relative to CD8+ T cells in bloodfrom uninfected WT mice. (B) Virus-specific CD8+ T cell frequencies andabsolute numbers were enumerated in spleen at 8-days post infection(dpi). (C) FACS plots of virus-specific CD8+ T cell frequencies onGP₃₃₋₄₁+CD8+ T cells. (D) Frequencies and absolute numbers ofvirus-specific GP₆₆₋₇₆+CD4+ T cells in spleen 8-dpi. (E) FACS plots ofvirus-specific CD8+ T cell frequencies on virus-specific GP₆₆₋₇₆+CD4+ Tcells.

FIGS. 2A-E show that virus-specific PSGL-1 KO T cells display enhancedsurvival but not proliferation. WT mice are represented by black barsand PSGL-1 KO mice are represented by white bars. (A-B) Levels ofGP₃₃₋₄₁+CD4+ cells (A) and GP₆₆₋₇₆+CD4+ cells (B) from WT and PSGL-1spleens isolated at 8-dpi. (C-D) Levels of IL7Rα (C) and Bcl-2 (D) fromWT and PSGL-1 spleens isolated at 10-dpi (E) Levels of CD25 from WT andPSGL-1 blood at 10-dpi.

FIGS. 3A-G show enhanced effector T cell function in PSGL-1 KO mice. WTmice are represented by black bars and PSGL-1 KO mice are represented bywhite bars. (A) production of INF-γ and INF-γ+TNF-α in GP₃₃₋₄₁+CD8+cells. (B) production of INF-γ and INF-γ+TNF-α in NP₃₉₆₋₄₀₄+CD8+ cells.(C) production of CD107+INF-γ+Tet+ cells. (D) production of Granzyme Bin GP₃₃₋₄₁+CD8+ cells. (E) production of T-bet in GP₃₃₋₄₁+CD8+ cells.(F) production of Eomes in GP₃₃₋₄₁+CD8+ cells. (G) production of INF-γ,INF-γ+TNF-α and INF-γ+TNF-α+IL-2 in GP₆₆₋₇₆+CD4+ cells.

FIGS. 4A-D show inhibitory receptor expression on virus-specific Tcells. WT mice are represented by black bars and PSGL-1 KO mice arerepresented by white bars. (A) levels of PD-1 on virus specificGP₆₆₋₇₆+CD4+ T cells 7 dpi (B) levels of PD-1 on virus specificGP₆₆₋₇₆+CD4+ T cells 8 dpi (C) levels of PD-1 on virus specificGP₃₃₋₄₁+CD8+ cells and NP₃₉₆₋₄₀₄+CD8+ cells 8 dpi (D) levels of CD160and BTLA on virus specific GP₃₃₋₄₁+CD8+ cells.

FIGS. 5A-G show that the accumulation of PSGL-1 KO virus-specific CD8⁺and CD4⁺ T cells is cell intrinsic and PSGL-1 ligation of exhausted CD8⁺T cells diminished their survival. WT and PSG-L-1 KO naive P14transgenic T cells are represented by circles or 1PSGL-1 KO naive Smartatransgenic CD4⁺ T cells are represented by squares. (A-B) ratio ofPSGL-1 KO to WT in spleen (A) or lung (B) from WT and PSG-L-1 KO naiveP14 transgenic T cells (circles) or 1PSGL-1 KO naïve Smarta transgenicCD4⁺ T cells (squares) 1 dpi (C-D) the number of WT (circles) and PSGL-1KO (squares) P14 cells (C) or Smarta cells (D) in spleen 1 dpi (E) CFSEdilution in WT or PSGL-1 KO P14 cells at 2-dpi (F-G) frequency ofpropidium negative GP₃₃₋₄₁+CD8+ T cells (F) and PD-1 levels onGP₃₃₋₄₁+CD8+ T cells (G) from splenocytes isolated at 9-dpi.

FIGS. 6A-E show that PSGL-1 KO mice have accelerated viral control andextensive immunopathology. (A-C) serum LCMV viral levels (A), survivalcurves (B) and serum kinase levels (C) from WT mice represented by blackcircles or black bars and PSGL-1 KO mice represented by white squares orwhite bars at 8-dpi (D) H&E histology of uninfected and day 8 infectedWT and PSGL-1 KO lungs. (E) Pathology scores in lungs for WT and PSGL-1KO uninfected and 8-dpi.

FIGS. 7A-E show that optimal virus-specific CD8+ T cell function inPSGL-1 KO mice depends on CD4+ T cell help. WT mice represented by blackbars or black circles, PSGL-1 KO mice represented by white bars or whitesquares and PSGL-1 KO CD4-depleted mice represented by gray bars or graytriangles. (A) absolute numbers of GP₆₆₋₇₆+CD4+ T cells in spleen. (B)frequencies of GP₃₃₋₄₁ and NP₃₉₆₋₄₀₄ CD8+ T cells in blood at 8-dpi (C)absolute number of cytokine producing GP₃₃₋₄₁CD8+ T cells in spleen at10-dpi (D) PD-1 levels on GP₃₃₋₄₁+ and NP₃₉₆₋₄₀₄+ specific CD8+ T cellsin spleen at day 10 dpi (E) survival and serum viral levels at 10-dpi.

FIGS. 8A-E show the kinetics of virus-specific CD8⁺ T cell responseafter Cl13 infection. WT (black squares or bars) or PSGL1-KO (whitesquares or bars) mice. (A-D) absolute number of GP₃₃₋₄₁+ orNP₃₉₆₋₄₀₄+CD8+ T cells at day 4, 6, and 8 dpi (A,B, C) and 30 dpi (D).(E) dot plots represent a representative mouse.

FIGS. 9A-D show that PSGL-1 KO virus-specific T cells effectivelymigrate and accumulate in lung. (A-D) cells from WT (black bars) orPSGL1-K0 (white bars) mice lungs at day 7.5 dpi stained withD^(b)GP₃₃₋₄₁ tetramers or D^(b)NP₃₉₆₋₄₀₄ tetramers (A-B) orIA^(b)-GP₆₆₋₇₆ (C-D).

FIGS. 10A-E show cytokine receptor levels on virus-specific T cells. WTmice are represented by black squares or bars and PSGL-1 KO mice arerepresented by white squares or bars. (A) levels of IL-7Rα on GP₃₃₋₄₁cells at 4, 6 and 8 dpi (B) levels of levels of IL-7Rα on NP₃₉₆₋₄₀₄cells at 4, 6 and 8 dpi (C) levels of IL-21 at 9 dpi (D) levels of CD122at 9 dpi (E) levels of IL-6R at 9 dpi.

FIGS. 11A-E show that increased cytokine levels are expressed byeffector T cells from PSGL-1 KO mice. WT mice are represented by blackbars and PSGL-1 KO mice are represented by white bars. (A-C) productionof INF-γ (A,C) and TNF-α (B) on CD8+ T cells at 10 dpi (D-E) productionof TNF-α (D) and IL-2 (E) on CD4+ T cells at 10 dpi

FIGS. 12A-E show inhibitory receptor expression on virus-specific Tcells. WT mice are represented by black bars and PSGL-1 KO mice arerepresented by white bars. (A-E) levels of PD-1, CD160, and BTLA onvirus-specific GP₃₃₋₄₁− and NP₃₉₆₋₄₀₄− CD8⁺ T cells at day 8 pd. inspleen (A-B), in blood at day 15 dpi (C), 30 dpi (D), and in spleen atday 112 dpi (E).

FIGS. 13A-E show pre-infection phenotype of naive WT and PSGL-1 KO P14cells and proliferation WT and PSGL-1 KO P14 cells. WT mice arerepresented by black bars and PSGL-1 KO transgenic P14+ Tg mice arerepresented by white bars. (A-B) spleens stained with CD44 and CD62L (A)and CD25, CD69, and CDI27 (B). (C) frequency of WT or PSGL-1 KO P14cells in spleen and lung at day 13 dpi (D) Brdu incorporation in spleenand lung at day 13 dpi (E) representative histograms in spleen.

FIGS. 14A-B show greater pathology in PSGL-1 KO mice. WT uninfected miceare represented by black circles, PSGL-1 KO uninfected mice arerepresented by white bars squares, WT infected mice are represented byblack triangles, PSGL-1 KO infected mice are represented by whitetriangles mice. (A) pathology scores for WT and PSGL-1 KO lung andliver. (B) stomach, small and large intestines isolated from WT andPSGL-1 KO mice at day 9 dpi.

FIGS. 15A-D show the accumulation of virus specific PSGL-1KO T cellsafter Cl13 infection. (A-D) Virus specific CD8+ T cells were enumeratedin blood at 8 dpi (A) and 30 dpi (B) post infection and in the spleen(C) and lung (D) at 8 dpi.

FIGS. 16A-C show enhanced anti-viral function of CD8+ T cells fromPSGL-1 KO. (A-C) spleen cells stimulated with the NP₃₉₆₋₄₀₄ or GP₃₃₋₄₁peptides and analyzed for IFN-γ and TNF-α (A, B) or CD107 (C).

FIGS. 17A-B show deceased inhibitory receptor expression of virusspecific CD8+ T cells. (A-B) Virus specific T cells were assessed fromlevels of PD-1 (A) and CD60 and BTLA (B).

FIGS. 18A-C show PSGL-1 KO CD4+ T cells display improved survival andfunction with decreased inhibitory receptor expression. (A-C) thefrequencies (A), cytokine responses (B) and PD-1 and BTLA levels (C)were measured on 8 dpi

FIGS. 19A-B show PSGL-1 KO mice clear chronic LCMV and have greatercirculating inflammatory cytokines. (A) virus titers post infection. (B)cytokine levels on 8 dpi.

FIGS. 20A-D show T cell survival and PD-1 are modulated by PGSL-1deficiency after response to virus infection and after PSGL-1 blockade.(A-D) Levels of NP₃₉₆₋₄₀₄ (A) and GP₃₃₋₄₁ (B) specific CD8+ T cellsanalyzed post infection. (C) virus specific CD8+ T cells levels of PD-1measured from WT and PSGL-1 KO mice. (D) PD-1 levels on IAV-specificNP₃₉₆₋₄₀₄ or LCMV specific NP₃₉₆₋₄₀₄ CD8+ T cells were measured on 8dpi.

FIGS. 21A-B show PSGL-1 and PD-1 expression by T cells in tumormicroenvironment. (A) frequency of CD4+ cells (left), their expressionof PSGL-1 (middle) and expression of PD-1 by PSGL-1lo cells versus highPSGL-1high cells (right). (B) frequency of CD8+ T cells (left), theirhigh PSGL-1 expression (middle) and heterogeneous expression of PD-1.

FIGS. 22A-B show PSGL-1 expression and PSGL-1 binding by CD8+ T cells inmelanoma+ ears. (A) expression of PD-1 and PSGL-1 in CD44^(hi) vs.CD44^(lo) CD8+ T cells. (B) frequency of CD8+ T cells that havefunctional binding of PSGL-1.

FIGS. 23A-B show CD3+ T cells in melanoma tumor, and the frequencies ofCD4+ Tregs and CD4+ nonTregs. (A) CD3⁺ T cells monocuclear cells. (B)PSGL-1+, PD-1+ (double positive) CD4+ non Tregs (effector T cells) andTregs with high frequency within the disrupted tumors by flow cytometry.

FIG. 24 shows the presence of immune cells in melanoma, and theirexpression of PSGL-1. (A) T cells (CD4+ non Tregs, Tregs, CD8+ cells),NK T cells, NK cells, dendritic cells (DCs, MHC II+, CD11c) myeloidderived suppressor cells (MDSC, Gr1+, CD11b) and macrophages (MΦ,F4/80+, CD11b+) identified within melanoma tumors. (B) these subsets ofimmune cells expressed PSGL-1.

FIGS. 25A-F show the anti-tumor response of PSGL-1-deficient micecompared to WT mice. (A) tumor volume and weight. (B) the frequency ofeffector CD8+ and CD4+ T cells. (C) the expression of PD-1 by CD8+ andCD4+ T cells. (D-F) cytokine production by CD8+ and CD4+ T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the seminal discovery that P-selectinglycoprotein ligand-1 (PSGL-1) modulates the immune system and immuneresponses. Specifically, the present invention provides PSGL-1 agonistsand antagonists which increase the survival of multifunctional T cellsand viral clearance. The present invention further provides methods oftreating infectious diseases, cancer and immune and inflammatorydiseases and disorders using a PSGL-1 modulator.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. The definitions set forth below are forunderstanding of the disclosure but shall in no way be considered tosupplant the understanding of the terms held by those of ordinary skillin the art.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Antibodies are usually heterotetrameric glycoproteins of about 150,000daltons, composed of two identical light (L) chains and two identicalheavy (H) chains. Each light chain is linked to a heavy chain by onecovalent disulfide bond, while the number of disulfide linkages variesamong the heavy chains of different immunoglobulin isotypes. Each heavyand light chain also has regularly spaced intrachain disulfide bridges.Each heavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains. Each light chain has a variable domain atone end (V_(L)) and a constant domain at its other end; the constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light- and heavy-chainvariable domains. Each variable region is comprised of three segmentscalled complementarity-determining regions (CDRs) or hypervariableregions and a more highly conserved portions of variable domains arecalled the framework region (FR). The variable domains of heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FRs and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies. The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The Fc region of an antibody is the tail region of an antibody thatinteracts with cell surface receptors and some proteins of thecomplement system. This property allows antibodies to activate theimmune system. In IgG, IgA and IgD antibody isotypes, the Fc region iscomposed of two identical protein fragments, derived from the second andthird constant domains of the antibody's two heavy chains; IgM and IgEFc regions contain three heavy chain constant domains (CH domains 2-4)in each polypeptide chain. The Fc regions of IgGs bear a highlyconserved N-glycosylation site. Glycosylation of the Fc fragment isessential for Fc receptor-mediated activity. The N-glycans attached tothis site are predominantly core-fucosylated diantennary structures ofthe complex type. In addition, small amounts of these N-glycans alsobear bisecting GlcNAc and α-2,6 linked sialic acid residues.

The term “antibody” as used herein refers to intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies, andantibody fragments so long as they exhibit the desired biologicalactivity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab)₂, andFv fragments; diabodies, tribodies and the like; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, each monoclonal antibodyis directed against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey are synthesized by the hybridoma culture, uncontaminated by otherimmunoglobulins. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by hybridomas, byrecombinant DNA methods or isolated from phage antibody libraries.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding.

The terms “fusion molecule” and “fusion protein” are usedinterchangeably and are meant to refer to a biologically activepolypeptide usually a HVEM or antibody and an effector molecule usuallya protein or peptide sequence covalently linked (i.e. fused) byrecombinant, chemical or other suitable method. If desired, the fusionmolecule can be fused at one or several sites through a peptide linkersequence. Alternatively, the peptide linker may be used to assist inconstruction of the fusion molecule. Specifically preferred fusionmolecules are fusion proteins. Generally fusion molecule also can becomprised of conjugate molecules.

Fc-Fusion proteins (also known as Fc chimeric fusion protein, Fc-Ig,Ig-based Chimeric Fusion protein and Fc-tag protein) are composed of theFc domain of IgG genetically linked to a peptide or protein of interest.Fc-Fusion proteins have become valuable reagents for in vivo and invitro research.

The Fc-fused binding partner can range from a single peptide, a ligandthat activates upon binding with a cell surface receptor, signalingmolecules, the extracellular domain of a receptor that is activated upondimerization or as a bait protein that is used to identify bindingpartners in a protein microarray.

One of the most valuable features of the Fc domain in vivo, is it candramatically prolong the plasma half-life of the protein of interest,which for bio-therapeutic drugs, results in an improved therapeuticefficacy; an attribute that has made Fc-Fusion proteins attractivebio-therapeutic agents.

As used herein, the terms “nucleic acids” or “nucleic acid sequences”refer to oligonucleotide, nucleotide, polynucleotide, or any fragment ofany of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) ofgenomic or synthetic origin which may be single-stranded ordouble-stranded; and can be a sense or antisense strand, or a peptidenucleic acid (PNA), or any DNA-like or RNA-like material, natural orsynthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g.,e.g., double stranded iRNAs, e.g., iRNPs), nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably herein and refer to a compound of two or more subunitamino acids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g., phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. Full-length polypeptides, truncatedpolypeptides, point mutants, insertion mutants, splice variants,chimeric proteins, and fragments thereof are encompassed by thisdefinition. In various embodiments the polypeptides can have at least 10amino acids or at least 25, or at least 50 or at least 75 or at least100 or at least 125 or at least 150 or at least 175 or at least 200amino acids.

As used herein, the term “ small molecule” refers to a low molecularweight (<900 daltons) organic compound that may help regulate abiological process, with a size on the order of 10⁻⁹ m. Most drugs aresmall molecules. Small molecules can have a variety of biologicalfunctions, serving as cell signaling molecules, as drugs in medicine, aspesticides in farming, and in many other roles. These compounds can benatural (such as secondary metabolites) or artificial (such as antiviraldrugs); they may have a beneficial effect against a disease (such asdrugs) or may be detrimental (such as teratogens and carcinogens).Biopolymers such as nucleic acids and proteins, and polysaccharides(such as starch or cellulose) are not small molecules—though theirconstituent monomers-ribo- or deoxyribonucleotides, amino acids, andmonosaccharides, respectively-are often considered small molecules.

As used herein, the terms “treating” or “treatment” or “alleviation”refer to therapeutic treatment, prophylactic and/or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those in whom the disorder is to be prevented.

The term “therapeutic agent” as used herein includes a chemical compoundor composition capable of inducing a desired therapeutic effect whenadministered to a patient or subject. An example of a therapeutic agentof the present invention is an anti-PSGL-1 antibody or a PSGL-1 fusionprotein.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” of a drug used to treat a disease is an amount thatcan reduce the severity of a disease, reduce the severity of one or moresymptoms associated with the disease or its treatment, or delay theonset of more serious symptoms or a more serious disease that can occurwith some frequency following the treated condition. An “effectiveamount” may be determined empirically and in a routine manner, inrelation to the stated purpose.

The therapeutic agent may be administered by any suitable means,including topical, parenteral, subcutaneous, intraperitoneal,intrapulmonary, intranasal, intravenous, and/or intralesionaladministration in order to treat the subject.

As used herein, the term “T cell mediated disease or disorder” refers toany condition that would benefit from treatment with a PSGL-1 modulator.Examples of diseases and disorders that would benefit from anti-PSGL-1treatment include infectious diseases, cancer and immune, autoimmune andinflammatory diseases and disorders. In particular, subjects havingcancer or an infectious disease can benefit from PSGL-1 antagonisttreatment and subjects having an autoimmune or inflammatory disease ordisorder can benefit from PSGL-1 agonist treatment.

An infection occurs when an organism's body is invaded by pathogenicviruses, infectious virus particles (virions), fungus or bacteria thatcan attach to and enter susceptible cells. A vast number of virusescause infectious diseases. Examples of infectious diseases include, butare not limited to, Botulism, Bubonic plague, Calicivirus infection(Norovirus and Sapovirus), Chickenpox, Chlamydia, Cholera, Clostridiumdifficile infection, Common cold (Acute viral rhinopharyngitis; Acutecoryza), Creutzfeldt-Jakob disease (CJD), Dengue fever, Diphtheria,Ebola hemorrhagic fever, Gonorrhea, Hand, foot and mouth disease (HFMD),Helicobacter pylori infection, Hepatitis A, Hepatitis B, Hepatitis C,Hepatitis D, Hepatitis E, Herpes simplex, human immunodeficiency virus(HIV), Human papillomavirus (HPV) infection, Epstein-Barr VirusInfectious Mononucleosis (Mono), Influenza (flu), Legionellosis(Legionnaires' disease), Leprosy, Lyme disease (Lyme borreliosis),Malaria, Marburg hemorrhagic fever (MHF), Measles, Middle Eastrespiratory syndrome (MERS), Meningitis, Mumps, Pertussis (Whoopingcough), Plague, Progressive multifocal leukoencephalopathy, Rabies,Rhinovirus infection, Rocky Mountain spotted fever (RMSF), Rubella,Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies,Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster),Smallpox (Variola), Syphilis, Tetanus (Lockjaw), Tuberculosis, TyphoidFever, Valley fever, Viral pneumonia, West Nile Fever, and Yellow fever.

Infectious diseases are commonly treated with anti-viral agents,anti-bacterial agents or anti-fungal agents. Anti-viral agents included,but are not limited to, Abacavir, Aciclovir, Acyclovir, Adefovir,Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Balavir,Cidofovir, Combivir, Dolutegravir, Darunavir, Delavirdine, Didanosine,Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir,Ecoliever, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet,Fusion inhibitor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine,Imiquimod, Indinavir, Inosine, Integrase inhibitor, Interferon type III,Interferon type II, Interferon type I, Interferon, Lamivudine,Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir,Nevirapine, Nexavir, Nucleoside analogues, Novir, Oseltamivir,Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril,Podophyllotoxin, Protease inhibitor, Raltegravir, Reverse transcriptaseinhibitor, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir,Sofosbuvir, Stavudine, Synergistic enhancer, Tea tree oil, Telaprevir,Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir,Tromantadine, Truvada, Valaciclovir, Valganciclovir, Vicriviroc,Vidarabine, Viramidine, Zalcitabine, Zanamivir and Zidovudine.

Anti-bacterial agents include, but are not limited to, Amikacin,Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin,Streptomycin, Spectinomycin(Bs), Geldanamycin, Herbimyci, Rifaximin,Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem,Cefadroxil, Cefazolin, Cefalotin or Cefalothin), Cefalexin, Cefaclor,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime),Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin,Vancomycin, Telavancin, Dalbavancin, Oritavanci, Clindamycin,Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin,Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin,Aztreonam, Furazolidone, Nitrofurantoin(Bs), Oxazolidinones(Bs),Linezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Penicillin, Penicillin, Piperacillin, PenicillinG, Temocillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide,Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine,Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine,Sulfisoxazole, Trimethoprim, Demeclocycline, Doxycycline, Minocycline,Oxytetracycline, Tetracycline, Streptomycin and Fosfomycin.

Anti-fungal agents include, but are not limited to, Amphotericin B,Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin,Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole,Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole,Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole,Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole,Posaconazole, Propiconazole, Ravuconazole, Terconazole, Voriconazole,Abafungin, Amorolfin, Butenafine, Naftifine, Terbinafine, Anidulafungin,Caspofungin, and Micafungin.

An immune disease or disorder is a dysfunction of the immune system.These disorders can be characterized in several different ways: by thecomponent(s) of the immune system affected; by whether the immune systemis overactive or underactive and by whether the condition is congenitalor acquired. Autoimmune diseases arise from an abnormal immune responseof the body against substances and tissues normally present in the body(autoimmunity). A major understanding of the underlying pathophysiologyof autoimmune diseases has been the application of genome wideassociation scans that have identified a striking degree of geneticsharing among the autoimmune diseases.

Autoimmune disorders include, but are not limited to, Acute disseminatedencephalomyelitis (ADEM), Addison's disease, Agammaglobulinemia,Alopecia areata, Amyotrophic lateral sclerosis (aka Lou Gehrig'sdisease), Ankylosing Spondylitis, Antiphospholipid syndrome,Antisynthetase syndrome, Atopic allergy, Atopic dermatitis, Autoimmuneaplastic anemia, Autoimmune cardiomyopathy, Autoimmune enteropathy,Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner eardisease, Autoimmune lymphoproliferative syndrome, Autoimmunepancreatitis, Autoimmune peripheral neuropathy, Autoimmune polyendocrinesyndrome, Autoimmune progesterone dermatitis, Autoimmunethrombocytopenic purpura, Autoimmune urticaria, Autoimmune uveitis, Balodisease/Balo concentric sclerosis, Behçet's disease, Berger's disease,Bickerstaff s encephalitis, Blau syndrome, Bullous pemphigoid, Cancer,Castleman's disease, Celiac disease, Chagas disease, Chronicinflammatory demyelinating polyneuropathy, Chronic inflammatorydemyelinating polyneuropathy, Chronic obstructive pulmonary disease,Chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome,Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease,Complement component 2 deficiency, Contact dermatitis, Cranialarteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome,Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease,Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1,Diffuse cutaneous systemic sclerosis, Discoid lupus erythematosus,Dressler's syndrome, Drug-induced lupus, Eczema, Endometriosis,Eosinophilic fasciitis, Eosinophilic gastroenteritis, Eosinophilicpneumonia, Epidermolysis bullosa acquisita, Erythema nodosum,Erythroblastosis fetalis, Essential mixed cryoglobulinemia, Evan'ssyndrome, Fibrodysplasia ossificans progressiva, Fibrosing alveolitis(or Idiopathic pulmonary fibrosis), Gastritis, Gastrointestinalpemphigoid, Glomerulonephritis, Goodpasture's syndrome, graft versushost disease, Graves' disease, Guillain-Barré syndrome, Hashimoto'sencephalopathy, Hashimoto's thyroiditis, Henoch-Schonlein purpura,Herpes gestationis aka Gestational Pemphigoid, Hidradenitis suppurativa,Hughes-Stovin syndrome, Hypogammaglobulinemi, Idiopathic inflammatorydemyelinating diseases, Idiopathic pulmonary fibrosis, Idiopathicthrombocytopenic purpura, IgA nephropathy, Inclusion body myositis,Interstitial cystitis, Juvenile idiopathic arthritis aka Juvenilerheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenicsyndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus,Linear IgA disease, Lupoid hepatitis aka Autoimmune hepatitis, Lupuserythematosus, Majeed syndrome, Microscopic colitis, Microscopicpolyangiitis, Miller-Fisher syndrome, Mixed connective tissue disease,Morphea, Mucha-Habermann disease aka Pityriasis lichenoides etvarioliformis acuta, Multiple sclerosis, Myasthenia gravis, Myositis,Ménière's disease, Narcolepsy, Neuromyelitis optica, Neuromyotonia,Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord'sthyroiditis, Palindromic rheumatism, PANDAS (pediatric autoimmuneneuropsychiatric disorders associated with streptococcus),Paraneoplastic cerebellar degeneration, Paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis,Parsonage-Turner syndrome, Pemphigus vulgaris, Perivenousencephalomyelitis, Pernicious anaemia, POEMS syndrome, Polyarteritisnodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis,Primary sclerosing cholangitis, Progressive inflammatory neuropathy,Psoriasis, Psoriatic arthritis, Pure red cell aplasia, Pyodermagangrenosum, Rasmussen's encephalitis, Raynaud phenomenon, Reiter'ssyndrome, Relapsing polychondritis, Restless leg syndrome,Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis,Sarcoidosis, Schizophrenia, Schmidt syndrome, Schnitzler syndrome,Scleritis, Scleroderma, Serum Sickness, Sjögren's syndrome,Spondyloarthropathy, Stiff person syndrome, Still's disease, Subacutebacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome,Sydenham chorea, Sympathetic ophthalmia, Systemic lupus erythematosus,Takayasu's arteritis, Temporal arteritis, Thrombocytopenia, Tolosa-Huntsyndrome, Transverse myelitis, Ulcerative colitis, Undifferentiatedspondyloarthropathy, Urticarial vasculitis, Vasculitis, Vitiligo,Wegener's granulomatosis.

Inflammatory disease are a large group of disorders that underlie a vastvariety of human diseases. The immune system is often involved withinflammatory disorders, demonstrated in both allergic reactions and somemyopathies, with many immune system disorders resulting in abnormalinflammation. Non-immune diseases with etiological origins ininflammatory processes include cancer, atherosclerosis, and ischaemicheart disease. A large variety of proteins are involved in inflammation,and any one of them is open to a genetic mutation which impairs orotherwise dysregulates the normal function and expression of thatprotein. Examples of disorders associated with inflammation include Acnevulgaris, Asthma, Autoimmune diseases, Celiac disease, Chronicprostatitis, Glomerulonephritis, Hypersensitivities, Inflammatory boweldiseases, Pelvic inflammatory disease, Reperfusion injury, Rheumatoidarthritis, Sarcoidosis, Transplant rejection, Vasculitis, Interstitialcystitis, Atherosclerosis, Allergies, Myopathies, leukocyte defects andcancer.

The term “immune modulator” as used herein refers to any therapeuticagent that modulates the immune system. Examples of immune modulatorsinclude eicosanoids, cytokines, prostaglandins, interleukins,chemokines, checkpoint regulators, TNF superfamily members, TNF receptorsuperfamily members and interferons. Specific examples of immunemodulators include PGI2, PGE2, PGF2, CCL14, CCL19, CCL20, CCL21, CCL25,CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10,IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13,IL15, IL17, IL17, INF-α, INF-β, INF-ε, INF-γ, G-CSF, TNF-α, CTLA, CD20,PD1, PD1L1, PD1L2, ICOS, CD200, CD52, LTα, LTαβ, LIGHT, CD27L, 41BBL,FasL, Ox40L, April, TL1A, CD30L, TRAIL, RANKL, BAFF, TWEAK, CD40L, EDA1,EDA2, APP, NGF, TNFR1, TNFR2, LTβR, HVEM, CD27, 4-1BB, Fas, Ox40, AITR,DR3, CD30, TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, BAFFR, TACI,BCMA, Fn14, CD40, EDAR XEDAR, DR6, DcR3, NGFR-p75, and Taj. Otherexamples of immune modulators include tocilizumab (Actemra), CDP870(Cimzia), enteracept (Enbrel), adalimumab (Humira), Kineret, abatacept(Orencia), infliximab (Remicade), rituzimab (Rituxan), golimumab(Simponi), Avonex, Rebif, ReciGen, Plegridy, Betaseron, Copaxone,Novatrone, natalizumab (Tysabri), fingolimod (Gilenya), teriflunomide(Aubagio), BG12, Tecfidera, and alemtuzumab (Campath, Lemtrada).

Cancer is a group of diseases involving abnormal cell growth with thepotential to invade or spread to other parts of the body. Exemplarycancers described by the national cancer institute include: AcuteLymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood;Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; AdrenocorticalCarcinoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies;Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, ChildhoodCerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; BladderCancer, Childhood; Bone Cancer, Osteosarcoma/Malignant FibrousHistiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; BrainTumor, Brain Stem Glioma, Childhood; Brain Tumor, CerebellarAstrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/MalignantGlioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor,Medulloblastoma, Childhood; Brain Tumor, Supratentorial PrimitiveNeuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway andHypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); BreastCancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; BreastCancer, Male; Bronchial Adenomas/Carcinoids, Childhood: Carcinoid Tumor,Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical;Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central NervousSystem Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; CerebralAstrocytoma/Malignant Glioma, Childhood; Cervical Cancer; ChildhoodCancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia;Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of TendonSheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-CellLymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer,Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Familyof Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal GermCell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, IntraocularMelanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric(Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; GastrointestinalCarcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ CellTumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational TrophoblasticTumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathwayand Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer;Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver)Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin'sLymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; HypopharyngealCancer; Hypothalamic and Visual Pathway Glioma, Childhood; IntraocularMelanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma;Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia,Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood;Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood;Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia,Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary);Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; LungCancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; LymphoblasticLeukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma,AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma,Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's;Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma,Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma,Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central NervousSystem; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; MalignantMesothelioma, Adult; Malignant Mesothelioma, Childhood; MalignantThymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular;Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous NeckCancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome,Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides;Myelodysplastic Syndromes; Myelogenous Leukemia, Chronic; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma;Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood;Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer;Oral Cancer, Childhood; Oral Cavity and Lip Cancer; OropharyngealCancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; OvarianCancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor;Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; PancreaticCancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus andNasal Cavity Cancer; Parathyroid Cancer; Penile Cancer;Pheochromocytoma; Pineal and Supratentorial Primitive NeuroectodermalTumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult;Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; RenalCell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis andUreter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma,Childhood; Salivary Gland Cancer; Salivary Gland'Cancer, Childhood;Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma(Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma,Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, SoftTissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood;Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell LungCancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft TissueSarcoma, Childhood; Squamous Neck Cancer with Occult Primary,Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer,Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood;T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood;Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood;Transitional Cell Cancer of the Renal Pelvis and Ureter; TrophoblasticTumor, Gestational; Unknown Primary Site, Cancer of, Childhood; UnusualCancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer;Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway andHypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor.

The term “chemotherapeutic agent” as used herein refers to anytherapeutic agent used to treat cancer. Examples of chemotherapeuticagents include, but are not limited to, Actinomycin, Azacitidine,Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine,Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone,Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib,Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan,Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine,panitumamab, Erbitux (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3,denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin(trastuzumab), Remicade (infliximab), rituximab, Synagis (palivizumab),Mylotarg (gemtuzumab oxogamicin), Raptiva (efalizumab), Tysabri(natalizumab), Zenapax (dacliximab), NeutroSpec (Technetium (99mTc)fanolesomab), tocilizumab, ProstaScint (Indium-Ill labeled CapromabPendetide), Bexxar (tositumomab), Zevalin (ibritumomab tiuxetan(IDEC-Y2B8) conjugated to yttrium 90), Xolair (omalizumab), MabThera(Rituximab), ReoPro (abciximab), MabCampath (alemtuzumab), Simulect(basiliximab), LeukoScan (sulesomab), CEA-Scan (arcitumomab), Verluma(nofetumomab), Panorex (Edrecolomab), alemtuzumab, CDP 870, andnatalizumab.

The immune system is a system of biological structures and processeswithin an organism that protects against disease. This system is adiffuse, complex network of interacting cells, cell products, andcell-forming tissues that protects the body from pathogens and otherforeign substances, destroys infected and malignant cells, and removescellular debris: the system includes the thymus, spleen, lymph nodes andlymph tissue, stem cells, white blood cells, antibodies, andlymphokines. B cells or B lymphocytes are a type of lymphocyte in thehumoral immunity of the adaptive immune system and are important forimmune surveillance. T cells or T lymphocytes are a type of lymphocytethat plays a central role in cell-mediated immunity. There are two majorsubtypes of T cells: the killer T cell and the helper T cell. Inaddition there are suppressor T cells which have a role in modulatingimmune response. Killer T cells only recognize antigens coupled to ClassI MHC molecules, while helper T cells only recognize antigens coupled toClass II MHC molecules. These two mechanisms of antigen presentationreflect the different roles of the two types of T cell. A third, minorsubtype are the γδ T cells that recognize intact antigens that are notbound to MHC receptors. In contrast, the B cell antigen-specificreceptor is an antibody molecule on the B cell surface, and recognizeswhole pathogens without any need for antigen processing. Each lineage ofB cell expresses a different antibody, so the complete set of B cellantigen receptors represent all the antibodies that the body canmanufacture.

P-selectin glycoprotein ligand-1 (PSGL-1) is expressed by hematopoieticcells, and is a highly conserved ligand for the selectin family ofadhesion molecules, P, E, and L, known for initiating cell migration.Selectins are part of the broader family of cell adhesion molecules.PSGL-1 can bind to all three members of the family but binds with thehighest affinity to P-selectin. PSGL-1, a heavily glycosylatedsialomucin expressed on most leukocytes, has dual function as a selectinligand for leukocyte rolling on vascular selectins expressed ininflammation and as a facilitator of resting T cell homing into lymphoidorgans. PSGL-1 has been found to play a role in other contexts such ashematopoietic stem cell homing to the bone marrow, progenitor homing tothymus, and T cell homing to SLOs through interactions with chemokines.PSGL-1 deficiency appears to influence CD8+ T cell homeostasis in atleast three different ways: 1) by interfering with lymph node entry of Tcells, thereby limiting access to homeostatic cytokines and otherprosurvival signals in the lymph node; 2) by prolonging lymph noderesidence time, thereby extending exposure of T cells to prosurvivalsignals therein; and 3) by increasing T cell sensitivity to cytokines.

Chronic viral infections represent an altered state of homeostasis, anintricate balance between host and pathogen where both survive, albeitat the expense of suppression of the host immune system. Although theadhesion molecule PSGL-1 is thought to function primarily in cellmigration, its expression, signaling capacity, and binding specificitysuggest additional roles during infection.

PSGL-1 is a previously unrecognized negative regulator of T cellresponses that is linked to expression levels of multiple immuneinhibitory receptors. Therefore, PSGL-1 has significant translationalpotential for treating immune and inflammatory disorders, includingcancer. It has been shown that PSGL-1 deficiency enables CD8+ T cells tomount greater effector responses to influenza and lymphocyticchoriomenengitis (LCMV) viruses in murine models, as measured byenhanced cytotoxicity, cytokine production, and viral clearance.Moreover, effector and memory T cells persist in elevated frequenciesdue to improved survival. In a chronic infection model with theLCMV-variant strain, Clone 13, PSGL-1-deficiency prevents chronicinfection. CD8+ T cells retain effector functions and fail to developthe hallmarks of exhaustion that include high expression of PD-1 as wellseveral other inhibitory receptors. CD8+ T cells with characteristics ofexhaustion are found in the blood, lymph nodes, and tumor-infiltratingcells of melanoma patients. Although clinical trials with anti-PD-L1,-PD-1, or -CTLA-4 have shown some remarkable efficacy, the concept thatblocking multiple receptors will have greater impact is suggested bystudies of T cells from melanoma patients. Since greater function ofPSGL-1 KO T cells is associated with lower levels of multiple inhibitoryreceptors, we propose that blocking PSGL-1 could have the necessaryattributes to improve T cell immune responses to melanoma. PSGL-1 isalso expressed by regulatory T cells (Tregs) and DCs, whose tolerogenicfunctions are lost with PSGL-1 deficiency. Thus, blocking PSGL-1 couldnot only enhance the responses of effector T cells but also limit theimmunosuppressive responses of Tregs and DCs.

As described in the examples below, it was established that PSGL-1unexpectedly plays a role as a regulator of T cell function that can beexploited by a chronic virus to keep effector T cell immunity in checkthus permitting a persistent infection but also limiting immune mediatedhost tissue damage. In PSGL-1-deficient hosts, the CD8⁺ and CD4⁺ T cellsfailed to acquire hallmarks of T cell exhaustion, and instead developedinto robust multifunctional effectors that were maintained at elevatedlevels and together promoted early viral clearance, outcomes thatmechanistically required CD4⁺ T cells. This effective anti-viralimmunity was linked to reduced expression of inhibitory receptors onboth CD8⁺ and CD4⁺ T cells, as well as increased systemic levels ofproinflammatory cytokines and immune pathology. In the absence of CD4⁺ Tcells, the CD8⁺ T cells in PSGL-1-deficient mice failed to down regulateinhibitory receptors, displayed functional exhaustion, and were unableto support viral clearance. Therefore, relieving PSGL-1-dependentinhibition of CD4⁺ cells is key to inducing an effective anti-viralresponse by CD8⁺ T cells. PSGL-1 also plays a T cell intrinsic role inlimiting CD8⁺ and CD4+ effector cell survival, that could alsocontribute to reduced viral control.

Persistent viral replication following Cl13 infection drives CD8⁺ T cellexhaustion and apoptosis by extinguishing production, availability, andresponses to the ye survival cytokines, IL-2, 1L-7, and 1L-21.Importantly, PSGL-1-deficiency was associated with increased andsustained expression of IL-7Rα as well as Bc1-2, suggesting thatenhanced IL-7Rα signaling provides one mechanism to improve effector Tcell survival. This conclusion is supported by the finding thattherapeutic administration of IL-7 expands virus-specific CD8⁺ T cells,increases their effector function, and prevents persisting Cl13infection. The increased expression of the high affinity IL-2 receptor,CD25, on PSGL-1-deficient CD8⁺ T cells, and greater availability of IL-2from CD4⁺ T cells could also underlie improved CD8⁺ as well as CD4⁺ Tcell survival and function. Previous studies showed that IL-2 increasesvirus-specific CD8⁺ T cells and reduces viral loads when administered toCl13 infected mice. Since the progressive loss of function by CD8⁺ Tcells can lead to elimination of high affinity clones by apoptosismediated by TGF-α that can be reversed by IL-2 and 1L-7, our findingssuggest that in combination, enhanced IL-7 and 1L-2 signals tovirus-specific CD8⁻⁺ T cells could contribute to the mechanisms ofsurvival. However, since elevated serum levels of IL-21 were detected atthe peak of the CD8⁺ T cell response in PSGL-1-deficient mice and IL-21sustains virus-specific CD8⁺ T cells during chronic LCMV infection, thisγc cytokine that is produced by CD4⁺ T cells as well as other cell typescould also contribute to the mechanism of virus-specific CD8⁺ T cellsurvival. It was also observed increased levels of IL-6, which isnecessary for the generation and function of T follicular helper cells(Tfh), thereby leading to improved antibody responses that contribute toultimate control of chronic LCMV. The results described below indicatethat in WT mice, PSGL-1 plays a central role in dampening the T cellresponse to Cl13 by limiting virus-specific CD8⁺ and CD4+ T cellsurvival responses to several pro-survival cytokines and by constrainingproduction of key cytokines implicated in CD4 T cell help.

Chronic viral infections maintain anti-viral T cell responses in a stateof dysfunction that is associated with several immune inhibitoryreceptors. It is striking that each of the receptors analyzed, PD-1,CD160 and BTLA, was down-modulated on PSGL-1-deficient CD8⁺ and CD4⁺effectors after Cl13 infection and the anti-viral response wasdramatically improved. The results imply that PSGL-1 is normally a keypart of a regulatory program that prevents excessive T cell responses.PSGL-1-deficiency had no apparent effect on resting T cells nor did itlead to inherent inflammatory responses; however, its absence results inimmunopathology and increased mortality after Cl13 infection. Thisunderscores the function of PSGL-1 in balancing excessive T cellresponses and host tissue destruction to persistent antigen.Mechanistically, it was found that ligating PSGL-1 during antigenstimulation led to reduced survival of exhausted CD8⁺ T cells andupregulated PD-1 on the remaining viable cells, indicating that PSGL-1signaling links to expression of this receptor.

Changes in transcriptional regulation that are usually associated withCD8⁺ T cell dysfunction include reduced expression of homes and enhancedT-bet, which favor effector differentiation during chronic infection asopposed to memory formation during acute infection. These transcriptionfactors have opposing roles in chronic LCMV infection with moreexhausted virus-specific CD8⁺ T cells expressing higher levels of homesand reduced T-bet levels. Since elevated T-bet expression represses PD-1expression, it is striking that PSGL-1 deficiency caused reduced homesand more T-bet, further supporting the concept that changes intranscriptional regulation and markers of exhaustion are linked toPSGL-1 expression.

High PSGL-1 expression can be found on many cells types, including DCsand Tregs where it has been associated with functional regulation,delineating tolerogenic DCs and the most suppressive Tregs. Therefore,PSGL-1 could play roles on other cells that indirectly contribute to Tcell dysfunction during chronic infection. However, our results indicatethat these effects, if occurring in the context of PSGL-1-deficiency,require the activity of CD4⁺ T cells to realize the regulatory activityof PSGL-1. PSGL-1-deficient mice have been reported to have a subtlephenotype and our data indicate that infection is necessary to inducethe profound PSGL-1-dependent inhibitory effects. Despite alteredtrafficking of pre-T cells to the thymus, and of naive CD4⁺ T cells tolymph nodes peripheral naïve T cells appear to be generated normally inthe absence of PSGL-1, although memory phenotype cells are somewhatincreased possibly due to greater turnover. Given the T cell intrinsiceffects of PSGL-1-deficiency on effector cell survival and the reversalof the enhanced anti-viral immunity with CD4⁺ T cell depletion, moresubtle aspects of regulation in the context of PSGL-1 deficiency do notappear to make a substantive contribution to the outcomes of this study.

Recent clinical trials with anti-PD-1/PD-L1 and/or CTLA-4 support theconcept that immunity to chronic virus or tumors can be improved byinterfering with inhibitory pathways. Furthermore, targetingcombinations of inhibitory receptors can lead to greater efficacy andsince multiple inhibitory receptors are decreased by PSGL-1, thisreceptor could be a novel target whose inhibition might improve T cellresponses in several clinical contexts.

In one embodiment, the present invention provides a method of treating aT cell mediated disease or disorder comprising administering aP-selectin glycoprotein ligand-1 (PSGL-1) modulator to a subject in needthereof. In one aspect, the PSGL-1 modulator is an agonist orantagonist. In another aspect, the T cell mediated disease or disorderis an infectious disease, cancer, an autoimmune disorder or aninflammatory disorder. In an additional aspect, the infectious diseaseis Botulism, Bubonic plague, Calicivirus infection (Norovirus andSapovirus), Chickenpox, Chlamydia, Cholera, Clostridium difficileinfection, Common cold (Acute viral rhinopharyngitis; Acute coryza),Creutzfeldt-Jakob disease (CJD), Dengue fever, Diphtheria, Ebolahemorrhagic fever, Gonorrhea, Hand, foot and mouth disease (HFMD),Helicobacter pylori infection, Hepatitis A, Hepatitis B, Hepatitis C,Hepatitis D, Hepatitis E, Herpes simplex, human immunodeficiency virus(HIV), Human papillomavirus (HPV) infection, Epstein-Barr VirusInfectious Mononucleosis (Mono), Influenza (flu), Legionellosis(Legionnaires' disease), Leprosy, Lyme disease (Lyme borreliosis),Malaria, Marburg hemorrhagic fever (MHF), Measles, Middle Eastrespiratory syndrome (MERS), Meningitis, Mumps, Pertussis (Whoopingcough), Plague, Progressive multifocal leukoencephalopathy, Rabies,Rhinovirus infection, Rocky Mountain spotted fever (RMSF), Rubella,Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies,Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster),Smallpox (Variola), Syphilis, Tetanus (Lockjaw), Tuberculosis, TyphoidFever, Valley fever, Viral pneumonia, West Nile Fever, or Yellow fever.In certain aspect, the cancer is prostate, colon, abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, or urogenital tract. In afurther aspect, the autoimmune disorder is Addison's disease,amyotrophic lateral sclerosis, Crohn's disease, Cushing's Syndrome,diabetes mellitus type 1, graft versus host disease, Graves' disease,Guillain-Barré syndrome, lupus erythematosus, multiple sclerosis,psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis,scleroderma, systemic lupus erythematosus, transplant rejection, orvasculitis. In a specific aspect, the PSGL-1 modulator is an agonist andthe T cell mediated disease or disorder is an autoimmune or inflammatorydisease or disorder. In certain aspects, the PSGL-1 modulator is anantagonist and the T cell mediated disease or disorder is cancer or aninfectious disease.

In one aspect, the PSGL-1 modulator is an antibody, a small molecule, aprotein, a fusion protein or a nucleic acid. In a specific aspect, theantibody is a monoclonal antibody, chimeric antibody, human antibody orhumanized antibody. In another aspect, CD4+ dependent CD8+ T cellresponse is increased. In an additional aspect, virus specific T cellsare increased. In another aspect, Treg and DC response is increased. Ina further aspect, expression of FoxP3, IL-10, TGF-β and/or MHC class IIis increased. In an aspect, CD8+ secretion of IFNγ, TNFα and CD107 isincreased. In other aspects, expression of PD-1, BTLA and CD160 isdecreased or decreased. In one aspect, expression of CD25 and T-bet isincreased. In another aspect, viral clearance is increased. In anadditional aspect, the method further comprises the administration of atherapeutic agent. In a further aspect, the therapeutic agent is animmune modulator, a chemotherapeutic agent, an anti-viral agent, ananti-bacterial agent or an anti-fungal agent.

In an additional embodiment, the present invention provides for a methodof eliciting a T cell response comprising administering a PSGL-1modulator to a subject in need thereof. In an aspect, the PSGL-1modulator is an agonist or antagonist. In another aspect, the T cellmediated disease or disorder is an infectious disease, cancer, anautoimmune disorder or an inflammatory disorder. In one aspect, thePSGL-1 modulator is an antibody, a small molecule, a protein, a fusionprotein or a nucleic acid. In a specific aspect, the antibody is amonoclonal antibody, chimeric antibody, human antibody or humanizedantibody. In another aspect, CD4+ dependent CD8+ T cell response isincreased. In an additional aspect, virus specific T cells areincreased. In another aspect, Treg and DC response is increased. In afurther aspect, expression of FoxP3, IL-10, TGF-β and/or MHC class II isincreased. In an aspect, CD8+ secretion of IFNγ, TNFα and CD107 isincreased. In other aspects, expression of PD-1, BTLA and CD160 isdecreased or increased. In one aspect, expression of CD25 and T-bet isincreased.

In a further embodiment, the present invention provides a method ofrestoring T cell function comprising administering a P-selectinglycoprotein ligand-1 (PSGL-1) modulator to a subject in need thereof.In an aspect, the PSGL-1 modulator is an agonist or antagonist. Inanother aspect, the T cell mediated disease or disorder is an infectiousdisease, cancer, an autoimmune disorder or an inflammatory disorder. Inone aspect, the PSGL-1 modulator is an antibody, a small molecule, aprotein, a fusion protein or a nucleic acid. In a specific aspect, theantibody is a monoclonal antibody, chimeric antibody, human antibody orhumanized antibody. In another aspect, CD4+ dependent CD8+ T cellresponse is increased. In an additional aspect, virus specific T cellsare increased. In another aspect, Treg and DC response is increased. Ina further aspect, expression of FoxP3, IL-10, TGF-β and/or MHC class IIis increased. In an aspect, CD8+ secretion of IFNγ, TNFα and CD107 isincreased. In other aspects, expression of PD-1, BTLA and CD160 isdecreased or increased. In one aspect, expression of CD25 and T-bet isincreased. In an additional aspect, the method further comprises theadministration of a therapeutic agent. In a further aspect, thetherapeutic agent is an immune modulator, a chemotherapeutic agent, ananti-viral agent, an anti-bacterial agent or an anti-fungal agent.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a P-selectin glycoprotein ligand-1 (PSGL-1)modulator and a pharmaceutical carrier. In one aspect, the PSGL-1modulator is an antibody, a small molecule, a protein, a fusion proteinor a nucleic acid. In an additional aspect, the antibody is a monoclonalantibody, chimeric antibody, human antibody or humanized antibody.

The invention in all its aspects is illustrated further in the followingExamples. The Examples do not, however, limit the scope of theinvention, which is defined by the appended claims.

EXAMPLES Example 1 PSGL-1 Expression is Increased on Virus-Specific CD8+T Cells and PSGL-1 Deficient Mice have an Accumulation of Virus-SpecificT Cells During Cl13 Infection

To study PSGL-1 in chronic viral infection, the LCMV Cl13 virus was usedwhich results in viremia to 90-dpi and detectable virus in brain andkidney to 200-days post infection (dpi). PSGL-I levels on CD8+ T cellsspecific for the GP₃₃₋₄₁+ LCMV epitope was first examined by tetramerstaining. Although. PSGL-1 is expressed by all T cells, the levels wereincreased on virus-specific compared to naïve CD8+ T cells (FIG. 1 a ).To examine contributions of PSGL-1 to the anti-viral response, WT orPSGL-1-deficient mice were infected with Cl13 and analyzed CD8+ T cellsspecific for the LCMV GP₃₃₋₄₁+ and NP₃₉₆₋₄₀₄+ epitopes. PSGL-1-deficientmice had greatly increased frequencies and numbers of GP₃₃₋₄₁+CD8+ Tcells at 8-dpi (FIG. 1 b ). The difference in CD8+ T cell accumulationwas not observed until after 4-dpi (FIG. 8 a-b ) and tetramer cells weremaintained through 30-dpi (FIG. 8 c-e ). Most impressive was thepreservation of NP₃₉₆₋₄₀₄+ T cells in PSGL-1-deficient mice (FIG. 1 b,c), as these cells are largely deleted by 30-dpi in WT mice (FIG. 8 c-e). It was also found that virus-specific GP₆₆₋₇₆+CD4+ T cells wereincreased in PSGL-1 -deficient mice (FIG. 1 d,e ). This difference wasobserved by 5-dpi (FIG. 7 a ).

Since engagement of PSGL-1 on T cells by E- and P-selectin onendothelium can be important for migration to sites of inflammation,CD8+ T cell accumulation in the blood and spleen in PSGL-1-deficientmice could result from impaired migration to peripheral sites. The lung,which represents an alternate site of infection that, unlike the spleen,requires adhesion receptor regulation for T cell entry was examined.Consistent with findings in the spleen, virus-specific CD8+ (FIG. 9 a,b) and CD4+ T cells (FIG. 9 c,d ) in the lungs of PSGL-1-deficient miceaccumulated to a greater extent than in WT mice. The results show thatin PSGL-1-deficient mice, virus-specific T cells are preserved instrikingly higher numbers than are WT cells.

Example 2 PSGL-1-Deficient CD8+ T Cells have Enhanced Survival

Enhanced proliferation and/or survival could account for theaccumulation of virus-specific T cells in PSGL-1-deficient mice. Toassess proliferation, in vivo BrdU incorporation by virus-specific CD8+T cells was analyzed at 8-dpi. BrdU labeled −50% of WT GP₃₃₋41+CD8+ Tcells, but only −25% PSGL-1-deficient GP₃₃₋₄₁+CD8+ T cells (FIG. 2 a ).Similarly, 2× more WT GP₆₆₋₇₆ CD4+ T cells incorporated BrdU thanPSGL-1-deficient CD4+ virus-specific cells (FIG. 2 b ). Since divisiondid not seem to account for greater numbers of PSGL-1-deficient CD8+ Tcells, the expression levels of the survival molecules, IL-7Rα and itsdownstream signaling target Bc1-2 by CD8+ effector cells was examined.At 10-dpi both GP₃₃₋₄₁+ and NP₃₉₆₋₄₀₄+ T cells from PSGL-1-deficientmice displayed increased levels of IL-7Rα (FIG. 2 c ) and Bc1-2 (FIG. 2d ) compared to WT cells. Furthermore, IL-7Rα levels on PSGL-1-deficientvirus-specific CD8+ T cells were higher than on WT cells throughout theexpansion phase (FIG. 10 a,b ). CD25 levels on GP₃₃₋₄₁+ and NP₃₉₆₋₄₀₄+ Tcells from PSGL-1-deficient mice were also increased (FIG. 2 e ),whereas receptors for other cytokines that can enhance T cell survivalwere similar or reduced, including IL-21R, CD122 and IL-6R (FIG. 10 c-e). Together, these findings indicate that the accumulation ofvirus-specific CD8+ T cells in PSGL-1-deficient mice was most likely aresult of enhanced survival.

Example 3 Virus-Specific CD4⁺ and CD8⁺ T Cells in PSGL-1-Deficient Miceare Multifunctional Effectors

Since chronic virus infection in WT mice leads to sequential loss of theCD8+ T cell capacity to produce IFN-γ, TNF-α, and IL-2, as well as toproduce more than one of these cytokines simultaneously, cytokineproduction was examined. Much higher frequencies of PSGL-1-deficientGP₃₃₋₄₁+ T cells secreting IFNγ and IFN-γ+TNF-α together were foundcompared to WT cells (FIG. 3 a ). NP₃₉₆₋₄₀₄+ T cell responses were alsofunctional (FIG. 3 b ). On a per cell basis, −10% of GP₃₃₋₄₁+ T cellsproduced IFN-γ in WT vs −70% in PSGL-1-deficient mice (FIG. 3 a ).Furthermore, virus-specific PSGL-1-deficient CD8+ T cells producedsignificantly higher levels of cytokines (FIG. 11 a,b ). Both GP₃₃₋₄₁+and NP₃₉₆₋₄₀₄+ PSGL-1-deficient CD8+ T cells had enhanced CD107a levels,indicating better cytotoxic degranulation, together with IFN-γ section(FIG. 3 c ), although WT and PSGL-1-deficient GP_(33-41+CD)8+ T cellsdid not differ with respect to levels of granzyme B (Gzrnb) protein(FIG. 3 d ).

Transcriptional programs differ in acute vs chronic LCMV infection.While T-bet is important in regulating CD8+ T cell effector and memorydifferentiation during acute infections, during Cl13 infection exhaustedCD8+ T cells have reduced T-bet expression, which acts to sustain PD-1levels. Since T-bet can directly bind the Pdcdl gene that encodes PD-1and represses its expression, T-bet levels were examined in virusspecific CD8+ T cells and found increased expression in GP₃₃₋₄₄+ T cellsin PSGL-1-deficient mice at 10-dpi compared to WT cells (FIG. 3 e ).Furthermore, high Eomes levels, which mark terminally differentiatedCD8+ T cells destined to die in WT Cl13 infected mice, were reduced inGP₃₃₋₄₁+ T cells in PSGL-1-deficient mice (FIG. 3 f ). Thus, while CD8+T cells in WT mice develop exhaustion during Cl13 infection, inPSGL-1-deficient mice they instead generate multifunctional effectors.

The importance of CD4+ T cells during persistent infection ishighlighted by the failure of. CD4+ T cell-depleted Cl13 infected miceto control viremia. Improved functionality with respect to cytokineproduction was also observed with GP₆₆₋₇₆+CD4+ cells (FIG. 3 g , FIG. 11c-e ). Not only did more virus-specific CD4+ T cells fromPSGL-1-deficient mice produce elevated IFN-γ, TNF-α, and IL-2 comparedto those from WT mice and these virus-specific CD4+ T cells weremultifunctional with increased numbers of double/triple cytokineproducers (FIG. 3 g ) that also produced higher levels of cytokines(FIG. 11 c-e ). These findings demonstrate that greater numbers offunctionally superior virus-specific CD4+ T effector cells develop inPSGL-1-deficient mice than in WT mice after Cl13 infection.

Example 4 PSGL-1-Deficient T Cells have Reduced Inhibitory ReceptorExpression

Since T cell exhaustion is in part a consequence of expression ofmultiple inhibitory molecules including PD-1, CD1 60, Lag-3, and BTLA,these receptors were examined on virus-specific T cells in WT andPSGL-1-deficient mice after infection. It was found that PD-1 levels onvirus-specific GP₆₆₋₇₆+CD4+ T cells began to decrease by 7-dpi inPSGL-1-deficient mice, and further decreased on day 8 (FIG. 4 a, b ). Atthis time, BTLA levels were also diminished on PSGL-1-deficient CD4 Tcells (FIG. 4 b ). Likewise, compared to WT, PSGL-1-deficient CD8+ Tcells expressed lower levels of PD-1, but similar levels of CD160 andBTLA at 8-dpi (FIG. 12 a,b ). PSGL-1-deficient virus-specific CD8+ Tcells showed diminished expression of all three receptors by 9-dpi (FIG.4 c,d ). Furthermore, PD-1 downregulation was sustained at 15-, 30-, and112-dpi (FIG. 12 c-e ). These findings indicate that although notcoordinately regulated, reduced inhibitory receptor levels arecorrelated with functionality in virus-specific CD4+ and CD8+ T cells inPSGL-1-deficient mice after Cl13 infection.

Example 5 The Accumulation of Virus-Specific PSGL-1-Deficient T Cells isCell-Intrinsic

To address which features of the improved response to Cl13 inPSGL-1-deficient mice were intrinsic to T cells, WT and PSGL-1-deficientTCR transgenic CD8+ P14 cells, which are specific for the GP₃₃₋₄₁epitope of LCMV, were used and transferred these cells to WT hosts. Theactivation state of WT and PSGL-1-deficient P14 cells was examineddirectly ex vivo and found similar expression levels of CD44 and CD62L(FIG. 13 a ), and of CD25, CD69, and IL-7Rα (FIG. 13 b ). Then WT andPSGL-1-deficient P14 cells were co-transfected at a 1:1 ratio into WThosts and infected the mice with Cl13 one day later. An increased ratioof PSGL-1-deficient P14 cells to WT P14 cells was found in the spleen(FIG. 5 a ) and lung (FIG. 5 b ) at 5- and 7-dpi. This increase was alsoreflected by greater numbers of PSGL-1-deficient P14 cells in spleens(FIG. 5 c ) and increased frequencies in the spleen and lungs at 13-dpi(FIG. 13 c ). This accumulation was not a result of increasedproliferation as measured by BrdU incorporation at 8-dpi (data notshown) and 13-dpi (FIG. 13 d,e ), supporting the concept thatPSGL-1-deficient T cells survived better than WT cells. Furthermore,CFSE dilution in WT and PSGL-1-deficient P14 cells was identical at2-dpi, in fact at this time WT cells had a slight accumulation advantage(FIG. 5 e ). Despite the greater accumulation, PSGL-1-deficient cells,not unexpectedly, displayed an exhausted phenotype (decreased cytokineproduction and high PD-1) when responding in the WT environment withCl13 infection (data not shown). Thus, although the accumulation ofPSGL-1-deficient virus-specific CD8+ T cell was cell-intrinsic, theirfunctional restoration is dependent on additional factors in thePSGL-1-deficient environment. Next, the function of PSGL-1 invirus-specific CD4+ cells was examined with WT and PSGL-1-deficienttransgenic Smarta CD4+ T cells. When co-transferred to WT mice, anincreased ratio of PSGL-1-deficient to WT cells was observed on 5- and7-dpi in spleen (FIG. 5 a ) and lungs (FIG. 5 b ) after infection, withparallel increased numbers (FIG. 5 d ). Despite having a cell-intrinsicsurvival, both CD8+ and CD4+ PSGL-1-deficient effectors fail to befunctionally rescued in a WT environment.

Example 6 PSGL-1 Ligation Decreases Survival of Exhausted CD8⁺ T Cells

To determine the impact of PSGL-1 ligation on virus-specific CD8+ Tcells, splenocytes from WT mice were isolated at 9-dpi, a point whenCD8+ T cells are functionally exhausted. A −7% viable tetrarner+CD8+ Tcells was found after 4 days of GP₃₃₋₄₁ stimulation (FIG. 5 f ). Whensplenocytes were stimulated with GP₃₃₋₄₁ peptide in the presence ofanti-PSGL-1 antibody, tetramer CD8+ T cells survival decreased by −50%,to levels similar to those in cultures with media containing only IgG oranti-PSGL-1 (FIG. 5 f ). PD-1 levels were increased after peptidestimulation, and these levels were further elevated when, anti-PSGL-1was present during peptide stimulation (FIG. 5 g ). These results showthat PSGL-1 ligation during antigen stimulation limits the survival ofvirus-specific CD8+ T cells and can enhance their PD-1. expression.

Example 7 PSGL-1-Deficient Mice Clear Chronic LCMV Control but ShowEnhanced Immunopathology

Since improved functional CD4+ and CD8+ T cell responses together withdecreased immune inhibitory receptor expression was observed inPSGL-1-deficient mice compared to WT, the ability of PSGL1-deficientmice to control the Cl13 virus was examined. It was found that comparedto WT mice, which had elevated viremia to 30-dpi, PSGL-1-deficient micecleared the virus from blood by 15-dpi (FIG. 6 a ). However, theimproved anti-viral T cell response was associated with >50% mortality,with death beginning at 8-dpi (FIG. 6 b ). To address systemicinflammation, proinflammatory cytokines were examined and observed thatPSGL-1-deficient mice had dramatically elevated serum levels of IL-6,IL-21, TNF-α, and IFN-γ (FIG. 6 c ). Furthermore, lung immunopathologywhich included lung infiltrates (FIG. 6 d , FIG. 14 a ), edema, andinflammation at 8-dpi (FIG. 6 d,e ) was increased in PSGL-1-deficientinfected mice. Elevated pathology was also evident in the kidneys,livers, and intestines (FIG. 14 a,b ). Thus, although PSGL-1-deficientmice controlled viral replication much more effectively than WT mice,this results in extensive inflammation and increased mortality due toimmunopathology. Therefore, PSGL-1 functions to limit an overlyexuberant effector response.

Example 8 Optimal Virus-Specific CD8⁺ T Cell Function inPSGL-1-Deficient Mice Requires CD4⁺ T Cells

Increases in GP₆₆₋₇₆+CD4+ T cells were observed in PSGL-1-deficient miceby 5-dpi and with further increasing accumulation to 9-dpi (FIG. 7 a ),consistent with a role for. PSGL-1 deficiency in CD4+ T cells inimpacting CD8 T cell response. To examine their contribution, CD4+ Tcells from PSGL-1-deficient mice were depleted by administering anti-CD4antibody. The CD8+ T cell responses to Cl13 were compared to those ofPSGL-1-deficient CD8+ T cells from mice treated with a control antibody,or of WT T cells. It was found that CD4+ T cell-depletedPSGL-1-deficient mice had reduced frequencies of GP₃₃₋₄₁+ andNP₃₉₆₋₄₀₄+CD8+ T cells to levels found in WT mice (FIG. 7 b ).Furthermore, they had reduced numbers of IFN-γ and IFN-γ+TNF-α+CD8+ Tcells compared to PSGL-1-deficient mice that had CD4+ T cells at 10-dpi(FIG. 7 c ). This was mirrored by changes in PD-1 levels on GP₃₃₋₄₁+ andNP₃₉₆₋₄₀₄+ T cells, which remained elevated and were to similar levelson WT cells (FIG. 7 d ). Unlike PSGL-1-deficient mice in which serumCl13 levels were reduced at 10-dpi, in CD4+ T cell depleted mice, thevirus levels remained comparable to those in WT mice (FIG. 7 e ),whereas the titers in PSGL-1-deficient mice were decreasing at this timeFurthermore, mortality of PSGL-1-deficient mice after 10-dpi wasprevented in CD4+ T cell depleted mice (FIG. 7 e ). These results showthat for virus-specific PSGL-1-deficient CD8+ T cells to escapefunctional exhaustion, they require help from CD4+ T cells. Thus bytheir improved numbers and function, it was determined that CD4+ T cellsmodulate CD8+ T cell function in the context of PSGL-1 deficiency.

Example 9 Materials & Methods

Mice. C57BL/6.1 mice and Selplg−/− mice were purchased from the Jacksonlaboratories. Mice were bred and maintained in specific pathogen-freefacilities and were infected in conventional BSL-2 facilities at theSanford-Burnham Medical Research Institute. Selplg−/− mice werebackcrossed to C57BL/6J mice for more than 10 generations. PI4 andSmarta TCR transgenic mice were obtained from Charles D. Surh (TheScripps Research Institute). These mice were bred to Ly5.1(B6.SJL-Ptprc^(a) Pepc^(b)/BoyJ) mice and to Thy1.1 (B6.PL-Thyl^(a)/CyJ), Selplg−/− mice. Mice used for experiments were at least sixweeks of age. Experiments were in compliance with the Sanford-BurnhamMedical Research institute IACUC regulations and veterinarian-approved.

Infection, proliferation, and cell transfer. LCMV Cl13 strain waspropagated in baby hamster kidney cells and titrated on Vero Africangreen monkey kidney cells. Frozen stocks were diluted M Vero cell mediaand 2×10⁶ plaque-forming units (PFU) of LCMV Cl13 were injected i.v. Toassess proliferation mice were injected i.p. with 2 mg of BrdU(Sigma-Aldrich) 16 hr before isolating lymphocytes from the spleens andlungs at days 8 or 13 after infection. For adoptive transfer, WT P14 andPSGL-1 KO P14 cells were purified (Stemcell Tech) and 1×10³ cells weretransferred i.v. into WT mice that were infected with LCMV C113, 1 daylater.

Flow cytometry and staining. Cells were surface stained for 20 min at 4°C. or 1 hr 15 min at room temperature for tetramer staining in PBSsupplemented with FACS staining buffer (2% FBS and 0.01% sodium azide).The following antibodies from Biolegend were used: anti-CD8α (clone53-6.7), anti-PD-1 (clone RMP1-30), anti-CD4 (clone GKI.5), anti-CD90.1(clone OX-7), anti-CD45.1 (clone A20), anti-IL-21R (clone 4A9),anti-CD126 (clone D7715A7), anti-CD44 (clone IM7), anti-CD62L (cloneMEL-14). The following clones were from eBioscience: anti-CD127 (IL-7Rαclone A7R34), anti-CD160 (clone ebiocnx-3), anti-BTLA (clone 6f7). Thefollowing antibodies were from BD: anti-CD122 (clone TM-(31), anti-BcI-2(clone 3F11), anti-CD107 (clone 1D4B), anti-CD162 (clone 2PH1),anti-CD25 (clone 3C7), anti-CD69 (clone H12F3), anti-Va2 (clone B20.1).The H-2D^(b)-GP₃₃₋₄₁, H-2D^(b)-NP₃₉₆₋₄₀₄ tetramers were purchased fromBeckman Coulter. The 1A^(b)-₆₆₋₇₇ tetramer was provided by the NIH corefacility. Cells were washed twice with FACS staining buffer and fixedfor 15 min with 1% formaldehyde in PBS. Cells were washed twice, andresuspended in FACS staining buffer. For intracellular cytokinestaining, cells were fixed and perineabilized with the Cytofix/CytopermKit (BD) and were stained with anti-GzmB (clone MHGBO5: invitrogen),anti-TNF-α (clone MP6-XT22: biolegend), anti-IFN-γ (clone XIVIG1.2:Biolegend), anti-IL-2 (clone JES6-5H4: eBioscience). BrdU staining wasperformed using a kit from eBioscience. For transcription factordetection, cells were fixed and permeabilized using the FoxP3 stainingkit (eBioscience) and stained with anti-Tbet (clone 4B10: Biolegend),anti-Eomes (clone Dan11mag: eBioscience). Stained cells were analyzed ona LSRFortessa flow cytometer (BD).

Ex vivo peptide stimulation. 2×10⁶ splenocytes from infected animalswere seeded into 96-well round-bottom plates. The cells were stimulatedin vita-o for 5 hrs at 37° C. with 2 pg/mL of GP₃₃₋₄₁, NP₃₉₆₋₄₀₄ orGP₆₁₋₈₀ peptides (AnaSpec), 50 U/mL IL-2 (NCI), and 1 μg/ml Brefeldin A(Sigma). Stimulated cells were then washed, surface stained, and thenstained intracellularly. Ex vivo PSGL-1 ligation. 2×10⁶ splenocytes fromday 9 LCMV Cl13 infected animals were seeded into 96-well round-bottomplates. The cells were stimulated in vitro for 4 days at 37° C. with 50U/mL IL-2 (NIH) in RPMI-1640 (Cellgro) media supplemented with 10 mMhepes (Cellgro), 1× MEM non-essential amino acids (Cellgro), 1 mM sodiumpyruvate (Cellgro) and 10% heat-inactivated FBS (Hyclone), and thefollowing conditions: 10 μg/mL rat IgG (JacksonImmuno Research), 10μg/mL anti-PSGL-1 (4RAIO BioXCell), 2 pg/mL of GP₃₃₋₄₁ (AnaSpec), oranti-PSGL-1 (4RA10 BioXCell)+2 μg/mL of GP₃₃₋₄₁ (AnaSpec). Culturedcells were then washed, and stained with propidium iodide (1 μg/mL) for10 min. at room temperature. Cells were washed and stained withanti-CD8αa, H-2D-Gp₃₃₋₄₁ tetramer (Beckman Coulter), anti-PD-1 for 1 hr15 min. at room temperature in FRCS staining buffer. Stained cells wereanalyzed on a LSRFortessa flow cytometer (BD) and live cells determinedby excluding PI+ cells.

CFSE labeling. CD8⁺ T cells were negatively enriched from spleens ofuninfected WT and PSGL-1 KO P14 TCR transgenic mice (Stemcell Tech).Equal numbers of purified WT and PSGL-1 KO P14 cells were pooled andlabeled with 5 μM CFSE (Life Technologies) at 37° C. for 10 minutes, andwashed with PBS. WT and PSGL-1 KO P14 cells were co-transferred at a 1:1ratio (1×10⁶ cells of each) i.v. into WT recipients. WT hosts wereinfected with 2×10⁶ PFU LCMV Cl 13 and CFSE dilution examined by FACS atday 2 dpi.

CD4 depletion. Mice received two 500 μg intraperitoneal injections ofCD4-depleting antibody (clone GM 0.5, BioXCell) at day −1 and 0, andthen infected with LCMV Cl13. Efficacy of CD4 depletion was confirmed inblood and spleens of treated mice.

Cytokines and pathology. Serum was isolated from WT and PSGL-1 KO miceat day 8 dpi and cytokine levels for IL-6, IL-21, TNF-α, and IFN-γ wereexamined using a multiplex 9-bead custom cytokine array (Millipore).Samples were analyzed on a Luminex IS200 instrument. Liver, kidney, andlungs were fixed in zinc formalin (z-fix Anatech), embedded in paraffin,and sectioned. Tissues were stained with H&E and digitally scanned usingAperio ScanScope. Pathology scoring was a blind assessment of tissuesamples by a pathologist. Scores ranged from 0-3.5, with zero indicatingno pathology and greater scores indicated increased pathology.

Data Analysis. Flow cytometry data were analyzed using FlowJo software(TreeStar). Graphs were prepared using GraphPad Prism software.

Statistical Analysis. GraphPad. Prism software was used to analyzeexperimental groups using a student t test, significance was set top<0.05. A Mantel-Cox and Gehan-Breslow-Wilcoxon test was used to comparesurvival curves.

Example 10 Anti-Viral Responses

In studies of the LCMV Cl13 virus that produces a chronic infection inmice, it was discovered PSGL-1 deficiency resulted in increased numbersof virus-specific CD8+ T cells after infection (FIG. 15A,B), thatextended at least to d30 (FIG. 15C), indicating a lasting impact on Tcells persistence. This finding is noteworthy for NP₃₉₆₋₄₀₄ Specific Tcells which are deleted after Cl13 infection. This effect was due toimproved T cell survival rather than increased expansion (not shown).Since PSGL-1 regulates leukocyte homing and LCMV infection is systemic,T cell recruitment to a peripheral site was tested, in this case thelungs, to see if there was an effect. However, there were greaternumbers of virus-specific T cells in PSGL-1 KO than in WT mice (FIG.15D) ruling out a general impairment of migratory capacity. CD8+ T cellsprogressively lose effector function after Cl13 infection, first theability produce IL-2, TNF-α, and finally IFN-γ and CTL activity. Ineffective anti-viral responses, CD8+ T cells express multiple effectorfunctions simultaneously. As shown in FIG. 16 , both GP₃₃₋₄₁− andNP₃₉₆₋₄₀₄-specific T cells from PSGL-1 KO mice exhibited higherfrequencies of IFN-γ+ and IFN-γ/INF-α double+ cells than virus-specificCD8+ T cells from WT mice (FIG. 16A,B), indicating greaterfunctionality. Cytotoxic degranulation in combination with IFN-γproduction was also greater (FIG. 16C). The data demonstrate that PSGL-1has an unexpected dampening effect on the CD8+ T cell responses.

Exhausted CD8+ T cells are characterized by expression of multipleimmune inhibitory receptors, notably PD-1, but also BTLA, CD160, LAG 3,TIM-3, and 2B4. It was found that reduced levels inhibitory receptors onCD8+ T cells was associated with improved function (FIG. 17 ) for bothGP₃₃₋₄₁ and N_(P396-404) specific T cells. Lower levels of 2B4 and LAG 3were also found (not shown). CD4+ T cells responding to Cl13 in WT andPSGL-1 KO mice were analyzed. As shown for CD8+ T cells, there weregreatly increased frequencies of virus-specific CD4+ T cells in KO mice(FIG. 18A) that were associated with better survival. Furthermore,virus-specific CD4+ T cells from PSGL-1 KO mice produced more cytokinesthan those from WT mice, and there were more polyfunctional cells aswell (FIG. 18B). Greater function was associated with decreasedexpression of the inhibitory receptors, PD-1 and BTLA (FIG. 18C). Thesedata indicate that PSGL-1 has a broader function than mediatingmigration in T cells and acts as a repressor of both CD8+ and CD4+ Tcell responses. To determine whether improved T cell function andsurvival impacted the anti-viral response, viral clearance was measured.As shown in FIG. 17A, PSGL-1 deficiency enabled the Cl13 virus to becleared. Greater levels of the cytokines IL-6, 1L-21, TNF-α, and IFN-γwere also detected in the sera of PSGL-1 KO mice compared to WT mice(FIG. 19B), thus providing a readout for an improved response. Tofurther study PSGL-1 in anti-viral immunity, it was analyzed whetherPD-1 expression levels after infection with the Armstrong LCMV strain,which is rapidly cleared by the immune system. Greater persistence ofCD8+ T cells in this setting (FIG. 20 ) with an earlier and morepronounced effect of PD-1 on NP₃₉₆₋₄₀₄ (FIG. 20A) than GP₃₃₋₄₁ (FIG.20B) virus specific CD8+ T cells was observed. For both clones, lowerlevels of PD-1 of PSGL-1 KO T cells were observed (FIG. 20C). To addresswhether levels of PD-1 on PSGL-1 can be targeted, an anti-PSGL-1blocking antibody (4RA10) or control IgG was administered to WT miceafter infection with influenza A virus (IAV) or with Cl13 LCMV. Theanti-PSGL-1 treated groups had increased frequencies of virus specificCD8+ T cells (not shown) and these cells expressed lower levels of PD-1than the controls (FIG. 20D). Together our findings indicate that PSGL-1has a previously unknown general role in down modulating T cellsresponses.

Example 11 BRAFV600E PTEN Melanoma Model

One of the best currently available murine models of aggressive humanmelanoma is an inducible genetic model that combines the BRAFV600Emutation, which is present in −65% of patients, with silencing of PTEN,a combination found in −20% of patients. Cre/Lox induction of BRAFV600Eand deletion of PTEN is temporarily controlled in melanocytes withactivation of a melanocyte-tyrosinanse promoter, Try:CreER, by localizedtreatment of the skin with tamoxifen. There is 100% penetrance withpigmented lesions developing by 2 wks and metastatic disease by 1 mo.CD3+ T cells and CD45+ mononuclear cells within the skin-associatedtumors were detected (data not shown). Tumors were analyzed by flowcytometry. Both CD4+ and CD8+ T cells were readily detected (FIG. 21A,B, left panels). PSGL-1 is expressed on −½ the CD4+ cells, and it isthese cells that have high PD-1 (FIG. 21A, middle, and right panels).The CD8+ T cells were exclusively PSGL-P, had mixed expression of PD-1(FIG. 21B). Furthermore, in melanoma+ ears, increased PD-1 and PSGL-1expression were detected on activated CD44+CD8+ T cells (FIG. 22A).These exhausted PD-1hi T cells also had functional PSGL-1 binding (FIG.22B). These findings indicate that tumor-infiltrating CD8+ T cells haveincreased PD-1, PSGL-1 and PSGL-1 binding activity . . . In additionalstudies, melanoma was induced in mice by Cre/Lox induction of theBRAFV600E mutation and deletion of PTEN in melanocytes with activationof a melanocyte-tyrosinase promoter, Tyr::CreER, by localized treatmentof the skin with tamoxifen. At 1 mo after treatment we observed CD3+ Tcells monocuclear cells within (FIG. 23A), and PSGL-1+, PD-1+ (doublepositive) CD4+ non Tregs (effector T cells) and Tregs with highfrequency within the disrupted tumors by flow cytometry (FIG. 23B). Bycomparison, few T cells expressed both molecules in the skin draining LNof either tumor bearing mice or non tumor bearing mice. CD3+ T cellswere found to be distributed throughout the melanoma (FIG. 23A), andboth CD4+ non Tregs as well as Tre were detectable and coexpressed PD-1and PSGL-1 (FIG. 23B). Additional studies of melanomas from these miceshow that immune cells can be readily distinguished by flow cytometry,including CD8+ and CD4+ T cells, Tregs, MDSCs and DCs, NK cells, NK Tcells, and macropphages (FIG. 24 ).

Example 12 To Identify PSGL-1 Expressing Cells and their Responseswithin Primary and Metastatic Melanoma

Immune cell changes with melanoma development: The kinetics of tumorgrowth will be established and the development of infiltrates by 4hydroxytamoxifen (4-HT) treatment of Tyr:CreER;Braf/Ptenlox/lox mice onthe flank at 6 wks of age. Immunohistochernistry will be used to assesstumors at weekly intervals once tumors are detected, focusing inparticular on CD44− and CD8+ T cells and their localization at the tumormargins vs infiltration into the tumors. For flow cytometry studies, theprimary melanoma and draining LN will be compared and changes assessedthat occur after metastasis to other skin sites, LN, and lungs. PSGL-1and inhibitory receptor expression (PD-1, Tim-3, Lag-1, BTLA, CTLA-4,and CD160) will be analyzed on CD8+, CD4+ and FoxP3+ Tregs in thetumors, draining lymph nodes and nondraining lymph nodes, blood, spleen,and lungs with time after 4-HT treatment. Studies of the B16 melanomamodel showed that tumor-specific CD84− T cells can be monitored usingMHC Class I tetramers loaded with the peptide gp100₂₅₋₃₃ (KVPRNQDWL) ofpmel-1, the mouse homologue of gp100₂₅₋₃₃ a structural component of thehuman melanosome matrix that is expressed by rnelanocytes as wellmalignant melanoma cells. This peptide is expressed by the melanomasinduced in the BRAFV600E PTEN melanoma model. Therefore, to studytumor-specific T cell responses commercially available pmel-tetramerswill be used. Whether there are changes in the frequencies ormelanoma-specific CD8+ T cells, their expression levels of PSGL-1 willbe addressed and whether they show differences from the overall CD8+ Tcell population in inhibitory receptor expression. Intracellularstaining (ICS) after anti-CD3 stimulation will be used to monitor thefunctions of CD4+ and CD8+ T cells by cytokine production (IL-2, IFN-γ,TNF-α), and pmel peptide restimulation will be used to assess cytokineproduction by tumor-specific CD8+ T cells. For CD8+ T cells. Cytotoxicactivity by granzyme B staining will be tested as well as bydegranulation by measuring CD107.

The immunosuppressive activity of Tregs by FoxP3 expression levels,which correlate with function (16), will be tested as well as 1L-10 andTGF-β production by ICS. The ratios of effector CD4+ and CD8+ T cells toTregs will be evaluated in the melanomas and draining LN to determinechanges that occur with time after the appearance of melanoma in theskin. Expansion of these populations will be examined by using BrdUadministration in the drinking water, initiating treatment at 1, 2, 3,or 4 wks after 4-HT treatment, analyzing T cells in the melanomas,lymphoid tissues, and blood. To further interrogate the tumormicroenvironment. The frequencies of DCs (MHC II+, CD11c+), MDSCs (Gr1+,CD11b+), and M4 (F4/80+, CD11b+) will be analyzed. To address functionin these populations, ICS will be used to analyze production of IL-12and 1L-10, which are associated with immunogenic vs tolerogenic DCs(17), and M1 vs M2 macrophages, respectively, whereas IL-10distinguishes MDSCs.

Example 13 To Determine whether Targeting PSGL-1 Can Delay or Controlthe Development of Melanoma and its Metastases

Impact of PSGL-1 on melanoma. Using a bone marrow chimera approach,whether PSGL-1 deficiency leads to changes in the functions of immunecells within the melanoma or in lymphoid compartment will be addressed.PSGL-1 deficiency will be created in hematopoietic cells by injectingPSGL-1 KO bone marrow into irradiated Tyr:CreER;Braf/Ptenloxilox miceand treat with 4-HT 8 wks later. Controls will receive WT bone marrow.Changes with respect to tumor growth and metastasis will be assessed,and in the frequencies and functions as well as inhibitory receptorexpression on immune cells in the tumor microenvironment, lymphoidcompartment, lungs and blood using histology and flow cytometry,focusing on the best readouts and time points defined in those studies.Evidence of autoimmunity/immunopathology by histology will be examinedsince deficiency or blocking of inhibitory receptors is associated withthe development of inflammation.

FIG. 25 shows enhanced anti-tumor T cell responses in PSGL-1-deficientmice. WT (black bars or black circles), and PSGL-1 KO (white bars orsquares) received a subcutaneous injection of melanoma cells (Yumm 1.5.Tumor weight on d14 and volume to day 12 post-injection) (A). Theabsolute number of effector CD8+ (left) and CD4+ (right) T cells pergram of tumor (B). PD-1 expression on effector T cells (C). Cyokineproduction by CD4+ (D) and CD8+ T cells (E). Representative cytokinestaining is shown in (F).

To address T cell intrinsic effects of PSGL-1 deficiency, two approacheswill be used. First, chimeras where PSGL-1 will be deleted exclusivelyin T cells will be generated for comparison to chimeras with allelicallymarked WT cells. To achieve this, lethally irradiatedTyr:CreER;Braf/Ptenlox/lox mice (Thy1.2, CD45.2) are reconstituted witha 80:20 ratio of TCR WS KO (CD45.2):PSGL-1 KO bone marrow cells (Thy1.1,CD45.2). The mice will be treated with 4-HT 8 wks later and thedevelopment and metastases of the tumors and responses of the T cellswill be evaluated. As a second approach, pmel Tg mice will be bred toPSGL-1 Thy1.1, CD45.2 KO mice CD8+ T cells isolated from these or WTpmel Tg mice (Thy1.1, CD45.2) will be injected into Tyr:CreER;Braf/Ptenlox/lox (Thy 1.2, Ly 5.2) in an optimized dose. As an alternative, wehave used a melanoma cell line, (Yumm 1.5) derived from an inducedprimary melanoma. Tumor cells were implanted s.c. in a dose of 5×10⁵(right flank) into WT or PSGL-1 deficient mice. In FIG. 24 , tumorgrowth was monitored by volume and weight (A), and the frequencies ofeffector CD8+ and CD4+ T cells were determined (B). PSGL-1 deficientmice had smaller tumors and contained greater numbers of T cells. The Tcells expressed lower levels of PD-1 (C) and were more functional asmeasured by cytokine production (D-F).

Immunotherapeutic inhibition PSGL-1 function. Whether blocking PSGL-1with an agonist mAb (4RA10) or a recombinant PSGL-1 Fc fusion proteincan control growth and/or metastases of melanoma with reversal of T celldysfunction will be determined. These reagents will be tested as atreatment of the primary melanoma as well as after metastasis hasoccurred. For the primary tumor, local injection of mAb into the tumorwill be compared with systemic treatment. For local treatment, astarting dose of 100 μg of anti-PSGL-1 or control IgG at the time ofprimary tumor detection will be used. For systemic treatment 300μg/mAb/dose will be injected i.p. with treatment every other day for 5days and will follow the animals tumor growth and skin metastases. At 1mo, the animals will be examined for pmel-tetramer+CD8+ T cells, theirsurface expression of inhibitory receptors, and their responses in theblood, spleen, LN, and, as indicated, tumors. The effects of initiatingsystemic mAb treatment will be examined at 1, 2, 3 or 4 wks afterdetection of the primary tumor to address whether anti-PSGL-1 can impactthe anti-tumor response with progressing disease. As a second strategyto block PSGL-1, PSGL-1 Fc will be administered with human Ig as thecontrol. The mAb and fusion protein levels will be assessed in the serumevery 3-4 days by ELISA to determine the 1/2 lives, and to addresswhether there are changes in T cell phenotype and/or responses with thedecay of these reagents. Levels of IL-6, IL-21, TNF-α, and IFN-γ will bemeasured in the sera at weekly intervals after the initiation of therapyto determine if changes in cytokine levels occur, and whether there isan association with improved anti-T cell tumor activity or tumorresolution.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1-30. (canceled)
 31. A method of treating a T-cell-mediated disease ordisorder comprising administering a P-selectin glycoprotein ligand-1(PSGL-1) antagonist to a subject in need thereof, wherein theT-cell-mediated disease or disorder is a cancer, wherein the PSGL-1antagonist increases a CD4+-T-cell-dependent CD8+ T-cell response, andwherein the CD4+ T-cell or CD8+ T-cell express PSGL-1.
 32. The method ofclaim 31, wherein the cancer is selected from the group consisting ofprostate, colon, abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital tract.
 33. The method of claim 31, wherein thePSGL-1 antagonist is an antibody, a small molecule, a protein, a fusionprotein or a nucleic acid.
 34. The method of claim 33, wherein theantibody is a monoclonal antibody, chimeric antibody, human antibody, orhumanized antibody.
 35. The method of claim 31, wherein expression ofFoxP3, IL-10, TGF-β, and/or MHC class II is increased.
 36. The method ofclaim 31, wherein secretion of IFNγ, TNFα, and CD107 from CD8+ T-cellsis increased, or wherein expression of CD25 and T-bet is increased. 37.The method of claim 31, wherein expression of PD-1, BTLA, and CD160 isdecreased or increased.
 38. A method of eliciting a T-cell responsecomprising administering a PSGL-1 antagonist to a subject in needthereof, wherein the PSGL-1 antagonist increases a CD4+-T-cell-dependentCD8+ T-cell response, and wherein the CD4+ T-cell or CD8+ T-cell expressPSGL-1.
 39. The method of claim 38, wherein the subject has a cancer.40. The method of claim 38, wherein the PSGL-1 antagonist is anantibody, a small molecule, a protein, a fusion protein or a nucleicacid.
 41. The method of claim 40, wherein the antibody is a monoclonalantibody, chimeric antibody, human antibody, or humanized antibody. 42.The method of claim 38, wherein expression of FoxP3, IL-10, TGF-β,and/or MHC class II is increased.
 43. The method of claim 38, whereinsecretion of IFNγ, TNFα, and CD107 from CD8+ T-cells is increased, orwherein expression of CD25 and T-bet is increased.
 44. The method ofclaim 38, wherein expression of PD-1, BTLA, and CD160 is decreased orincreased.
 45. A method of restoring T-cell function comprisingadministering a P-selectin glycoprotein ligand-1 (PSGL-1) antagonist toa subject in need thereof, wherein the PSGL-1 antagonist increases aCD4+ -T-cell dependent CD8+ T-cell response, and wherein the CD4+ T-cellor CD8+ T-cell express PSGL-1.
 46. The method of claim 45, wherein thesubject has a cancer.
 47. The method of claim 45, wherein the PSGL-1antagonist is an antibody, a small molecule, a protein, or a nucleicacid.
 48. The method of claim 47, wherein the antibody is a monoclonalantibody, chimeric antibody, human antibody, or humanized antibody. 49.The method of claim 45, wherein expression of FoxP3, IL-10, TGF-β,and/or MHC class II is increased, wherein secretion of IFNγ, TNFα, andCD107 from CD8+ T-cells is increased, or wherein expression of CD25 andT-bet is increased.
 50. The method of claim 45, wherein expression ofPD-1, BTLA, and CD160 is increased or decreased.